The long range objective of this research is to understand the cell surface receptor regulated functions of astrocytes. It is recognized that astrocytes express and respond to numerous neurotransmitters, neuromodulators and growth factors yet little is known about the mechanisms of response to these signals and in many cases the responses themselves. However it is increasingly recognized that astrocytes play a significant role in brain function throughout life. During development astrocytes guide neuronal migration. In adult brain, processes such as arousal, sleep-wakefulness, spreading depression, initiation of seizure activity include a significant astrocytic component. In the aging of damaged brain, gliosis is a major feature mediated by reactive astrocytes. Most brain tumors are derived from astrocytes or their progenitors. AIDS dementia appears to involve an astrocytic component. Understanding these features of brain physiology and pathophysiology requires an understanding of the responses of astrocytes to chemical signals. Current aims are to understand the significance of chemical mediator/second messenger stimulated phosphorylation of the cytoskeletal intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin. The experimental approach is to stimulate phosphorylation of these proteins in a line of intact cultured human astrocytoma cells (U-251 cells), to selectively extract and purify these proteins and to identify the specific amino acid residues that are phosphorylated. Site directed mutagenesis will then be used to replace these residues of GFAP with ones that either cannot be phosphorylated (e.g. alanine for serine) or ones that mimic the phosphorylated state (e.g. aspartate for serine). The site directed mutagenesis will be done in a mutant of the U-251 cells in which a null mutation has been introduced int the GFAP gene by gene targeting as a part of this project. Cells expressing the respective mutated forms of GFAP will then be studied in comparison to the parent U-251 cells and the cells carrying the null mutation in the GFAP gene, regarding their ability to express astrocyte specific functions such as induction of the astrocyte specific enzymes glutamine synthetase and monoamine oxidase B and the ability of astrocytes to send out processes in response to the presence of a neuron on their surface.